Albumin derivatives with therapeutic functions

ABSTRACT

Utilization of albumin as a stable plasma transporter with a therapeutic function that is derived from a membrane receptor. The present invention is exemplified by the description of new therapeutic agents that can be used in the treatment of Acquired Immunodeficiency Syndrome: hybrid macromolecules composed of albumin derivatives coupled to derivatives of the CD4 receptor having a normal or a higher affinity for the HIV-1 virus.

The present invention involves the utilization of albumin derivatives inthe fabrication of therapeutic agents that can be used in the treatmentof certain viral diseases and cancers. More precisely, this inventioninvolves hybrid macromolecules characterized by the fact that they carryeither the active domain of a receptor for a virus, or the active domainof a molecule which can bind to a virus, or the active domain of amolecule able to recognize the Fc fragment of immunoglobulins bound to avirus, or the active domain of a molecule able to bind a ligand thatintervenes in a pathologic process, coupled to albumin or a variant ofalbumin. In the text that follows, the terms albumin derivatives oralbumin variants are meant to designate all proteins with a high plasmahalf-life obtained by modification (mutation, deletion, and/or addition)via the techniques of genetic engineering of a gene encoding a givenisomorph of albumin, as well as all macromolecules with high plasmahalf-life obtained by the in vitro modification of the protein encodedby such genes. Such albumin derivatives can be used as pharmaceuticalsin antiviral treatment due to the high affinity of a virus or of animmunoglobulin bound to a virus for a site of fixation present on thealbumin derivative. They can be used as pharmaceuticals in the treatmentof certain cancers due to the affinity of a ligand, for example a growthfactor, for a site of fixation present on the albumin derivative,especially when such a ligand is associated with a particular membranereceptor whose amplification is correlated with a transforming phenotype(proto-oncogenes). It should be understood in the text that follows thatall functionally therapeutic albumin derivatives are designatedindifferently by the generic term of hybrid macromolecules withantiviral function, or hybrid macromolecules with anticancer function,or simply hybrid macromolecules. In particular, the present inventionconsists in the obtention of new therapeutic agents characterized by thecoupling, through chemical or genetic engineering techniques, of atleast two distinct funtions:

-   -   (i) a stable plasma transporter function provided by any albumin        variant, and in particular by human serum albumin (HSA). The        genes coding for HSA are highly polymorphic and more than 30        different genetic alleles have been reported (Weitkamp L. R et        al., Ann. Hum. Genet. 37 (1973) 219-226). The albumin molecule,        whose three-dimensional structure has been characterized by        X-ray diffraction (Carter D. C. et al. Science 244 (1989)        1195-1198), was chosen to provide the stable transporter        function because it is the most abundant plasma protein (40 g        per liter in humans), it has a high plasma half-life (14-20 days        in humans, Waldmann T. A., in “Albumin Structure, Function and        Uses”, Rosenoer V. M. et al. (eds), Pergamon Press,        Oxford, (1977) 255-275), and above all it has the advantage of        being devoid of enzymatic function, thus permitting its        therapeutic utilization at high doses.    -   (ii) an antiviral or anticancer therapeutic function. The        antiviral function is to serve as a decoy for the specific        binding of a virus, or as a decoy for the binding of a        virus-immunoglobulin complex. For example, the antiviral        function can be provided by all of part of a specific receptor        normally used by a virus for its propagation in the host        organism, or by any molecule capable of binding such a virus        with an affinity high enough to permit its utilization in vivo        as an antiviral agent. The antiviral function can also be        provided by all or part of a receptor capable of recognizing        immunoglobulins complexed with a virus, or by any molecule        capable of binding such complexes with an affinity high enough        to permit its utilization in vivo as an antiviral agent. The        anti-cancer function is to serve as a decoy for the binding of a        ligand and in particular a growth factor implicated in an        oncogenic process, and is provided by all or part of a cellular        proto-oncogene, or by any molecule capable of binding such a        ligand with an affinity high enough to allow its utilization in        vivo as an anticancer agent.    -   (iii) in cases where a high local concentration of the        therapeutic function is desirable, for example because it        synergizes an inhibition of the infectivity of a virus in vivo a        third function allowing the dimerization or the polymerization        of the therapeutically active hybrid macromolecule can be added,        possibly in a redundant fashion. For example, such a function        could be provided by a “leucine zipper” motif (Landschulz W. H.        et al., Science 240 (1988) 1759-1764), or by protein domains        known to be necessary for homodimerization of certain proteins        such as the domain of the product of the tat gene coded by the        HIV-1 viral genome (Frankel A. D. et al., Science 240 (1988)        70-73; Frankel A. D. et al., Proc. Natl. Acad. Sci. USA        85 (1988) 6297-6300).

In the present invention, the plasma transporter function, thetherapeutic function, and a potential polymerization function, areintegrated into the same macromolecule using the techniques of geneticengineering.

One of the goals of the present invention is to obtain hybridmacromolecules derived from HSA which can be useful in the fight againstcertain viral diseases, such as Acquired Immunodeficiency Syndrome(AIDS). Another goal is to obtain hybrid HSA macromolecular derivativesuseful in the treatment of certain cancers, notably those cancersassociated with genomic amplification and/or overexpression of humanproto-oncogenes, such as the proto-oncogene c-erbB-2 (Semba K. et al.,Proc. Natl. Acad. Sci. USA. 82 (1985) 6497-6501; Slamon D. J. et al.,Science 235 (1987) 177-182; Kraus M. H. et al., EMBO J. 6 (1987)605-610).

The HIV-1 virus is one of the retroviruses responsible for AcquiredImmunodeficiency Syndrome in man. This virus has been well studied overthe past five years; a fundamental discovery concerns the elucidation ofthe role of the CD4 (T4) molecule as the receptor of the HIV-1 virus(Dalgleish A. G. et al., Nature 312 (1984) 763-767; Klatzmann D. et al.,Nature 312 (1984) 767-768). The virus-receptor interaction occursthrough the highly specific binding of the viral envelope protein(gp120) to the CD4 molecule (McDougal et al., Science 231 (1986)382-385). The discovery of this interaction between the HIV-1 virus andcertain T lymphocytes was the basis of a patent claiming the utilizationof the T4 molecule or its antibodies as therapeutic agents against theHIV-1 virus (French patent application FR 2 570 278).

The cloning and the first version of the sequence of the gene encodinghuman CD4 has been described by Maddon et al. (Cell 42 (1985) 93-104),and a corrected version by Littmann et al. (Cell 55 (1988) 541): the CD4molecule is a member of the super-family of immunoglobulins andspecifically, it carries a V1 N-terminal domain which is substantiallyhomologous to the immunoglobulin heavy chain variable domain (Maddon P.J. et al., Cell 42 (1985) 93-104). Experiments involving in vitro DNArecombination, using the gene coding for the CD4 molecule, have provideddefinite proof that the product of the CD4 gene is the principalreceptor of the HIV-1 virus (Maddon P. J. et al., Cell 47 (1986)333-348). The sequence of this gene as well as its utilization as ananti-HIV-1 therapeutic agent are discussed in International patentapplication WO 88 013 040 A1.

The manipulation of the CD4 gene by the techniques of DNA recombinationhas provided a series of first generation soluble variants capable ofantiviral action in vitro (Smith D. H. et al., Science 238 (1987)1704-1707; Traunecker A. et al., Nature 331 (1988) 84-86; Fischer R. A.et al., Nature 331 (1988) 76-78; Hussey R. E. et al., Nature 331 (1988)78-81; Deen K. C. et al., Nature 331 (1988) 82-84), and in vivo(Watanabe M. et al., Nature 337 (1989) 267-270). In all cases, it wasobserved during various in vivo assays in animals (rabbit, monkey) aswell as during phase I clinical trials, that the first generationsoluble CD4 variant consisting of the CD4 molecule lacking the twodomains in the C-terminal region has a very short half-life:approximately 15 minutes in rabbits (Capon et al., Nature 337 (1989)525-531), while 50% of first generation soluble CD4 administeredintramuscularly to Rhesus monkeys remained bioavailable for 6 hours(Watanabe et al., Nature 337 (1989) 267-270). In addition, Phase Iclinical trials conducted on 60 patients presenting AIDS or ARC (“AidsRelated Complex”) indicated that the half-life of the Genentech productvaried between 60 minutes (intraveinous administration) and 9 hours(intramuscular administration) (AIDS/HIV Experimental TreatmentDirectory, AmFAR, May 1989). Clearly, a therapeutic agent with such aweak stability in vivo constitutes a major handicap. In effect, repeatedinjections of the product, which are costly and inconvenient for thepatient, or an administration of the product by perfusion, becomenecessary to attain an efficient concentration in plasma. It istherefore especially important to find derivatives of the CD4 moleculecharacterized by a much higher in vivo half-life.

The part of the CD4 molecule which interacts with the HIV-1 virus hasbeen localized to the N-terminal region, and in particular to the V1domain (Berger E. A. et al., Proc. Natl. Acad. Sci. USA 85 (1987)2357-2361). It has been observed that a significant proportion (about10%) of HIV-1-infected subjects develop an immune response against theCD4 receptor, with antibodies directed against the C-terminal region ofthe extra-cellular portion of the receptor (Thiriart C. et al., ADS 2(1988) 345-352; Chams V. et al., AIDS 2 (1988) 353-361). Therefore,according to a preferred embodiment of the present invention, only theN-terminal domains V1 or V1V2 of the CD4 molecule, which carry all theviral binding activity, will be used in fusion with the stabletransporter function derived from albumin.

On the basis of the homology observed with the variable domain ofimmunoglobulins, several laboratories have constructed genetic fusionsbetween the CD4 molecule and different types of immunoglobulins,generating hybrid immunoglobulins with antiviral action in vitro (CaponD. J. et al., Nature 337 (1989) 525-531; Traunecker A. et al., Nature339 (1989) 68-70; also see International patent application WO 8902922). However, the implication of the FcγRIII receptor (type 3receptor for the Fc region of IgG's), which in humans is the antigenCD16 (Unkeless J. C. and Jacquillat C., J. Immunol. Meth. 100 (1987)235-241), in the internalization of the HIV-1 virus (Homsy J. et al.,Science 244 (1989) 1357-1360) suggests an important role of thesereceptors in viral propagation in vivo. The receptor, which has beenrecently cloned (Simmons D. and Seed B., Nature 333 (1988) 568-570), ismainly located in the membranes of macrophages, polynuclear cells andgranulocytes, but in contrast to CD4, the CD16 receptor also exists in asoluble state in serum (Khayat D. et al., J. Immunol. 132 (1984)2496-2501; Khayat D. et al., J. Immunol. Meth. 100 (1987) 235-241). Itshould be noted that the membraneous CD16 receptor is used as a secondroute of entry by the HIV-1 virus to infect macrophages, due to thepresence of facilitating antibodies (Homsy J. et al., Science 244 (1989)1357-1360). This process of infection which involves an “Fc receptor” atthe surface of target cells (for example the CD16 receptor), and the Fcregion of antibodies directed against the virion, is named ADE(“Antibody Dependent Enhancement”); it has also been described for theflavivirus (Peiris J. S. M. et al., Nature 289 (1981) 189-191) and theVisna-Maedi ovine lentivirus (Jolly P. E. et al., J. Virol. 63 (1989)1811-1813). Other “Fc receptors” have been described for IgG's (FcγRIand FcγRII for example) as well as for other classes of immunoglobulins,and the ADE phenomenon also involves other types of “Fc receptors” suchas that recognized by the monoclonal antibody 3G8 (Homsy J. et al.,Science 244 (1989) 1357-1360; Takeda A. et al., Science 242 (1988)580-583). One can thus call into question the efficiency of hybridantiviral macromolecules which depend uniquely on fusions betweenimmunoglobulins and all or part of a receptor normally used by a virussuch as HIV-1 for its propagation in the host; in effect, the presenceof a functional Fc fragment on such molecules could actually facilitateviral infection of certain cell types. It is also important to obtainCD4 derivatives that can be used at high therapeutic concentrations.

A different type of chimeric construction involving the bacterialprotein MalE and the CD4 molecule has been studied (Clément J. M. etal., C.R. Acad. Sci. Paris 308 series m (1989) 401-406). Such a fusionallows one to take advantage of the properties of the MalE protein, inparticular regarding the production and/or purification of the hybridprotein. In addition, the construction of a genetic fusion between theCD4 molecule and a bacterial toxin has also been described (Chaudhary V.K. et al., Nature 335 (1988) 369-372). In these cases, utilization of agenetic fusion involving a bacterial protein for therapy in humans canbe questionable.

The discovery of the role of the ADE phenomenon in the propagation ofcertain viruses, in particular lentiviruses including HIV-1, justifiesthe search for alternatives to both the development of an anti-AIDSvaccine, and to the development of therapeutic agents based solely onfusions between immunoglobulins and molecules capable of binding thevirus. This is why the anti-AIDS therapeutic agents described in thepresent invention are based on the fusion of all or part of a receptorused directly or indirectly by the HIV-1 virus for its propagation invivo, with a stable plasma protein, devoid of enzymatic activity, andlacking the Fc fragment.

In particular, the present invention concerns the coupling, mainly bygenetic engineering, of human albumin variants with a binding site forthe HIV-1 virus. Such hybrid macromolecules derived from human serumalbumin are characterized by the presence of one or several variants ofthe CD4 receptor arising from the modification, particularly by in vitroDNA recombination techniques (mutation, deletion, and/or addition), ofthe N-terminal domain of the CD4 receptor, which is implicated in thespecific interaction of the HIV-1 virus with target cells. Such hybridmacromolecules circulating in the plasma represent stable decoys with anantiviral function, and will be designated by the generic term HSA-CD4.Another goal of this invention concerns the coupling of human albuminvariants with variants of the CD16 molecule, which is implicated in theinternalization of viruses including HIV-1 (to be designated by thegeneric term HSA-CD16), and in general the coupling of albumin variantswith molecules capable of mimicking the cellular receptors responsiblefor the ADE phenomenon of certain viruses, and in particular thelentiviruses.

The principles of the present invention can also be applied to otherreceptors used directly or indirectly by a human or animal virus for itspropagation in the host organism. For example:

-   -   1/ intercellular adhesion molecule 1 (ICAM-1), shown to be the        receptor for human rhinovirus HRV14 (Greve J. M. et al., Cell        56 (1989) 839-847; Staunton D. E. et al., Cell 56 (1989)        849-853);    -   2/ poliovirus receptor, recently cloned by Mendelsohn et al.        (Cell 56 (1989) 855-865);    -   3/ the receptor of complement factor C3D which is the receptor        of Epstein-Barr virus (EBV) in human cells (Fingeroth J. D. et        al., Proc. Natl. Acad. Sci. USA 81 (1984) 4510-4514), this virus        being responsible for infectious mononucleosis and for certain        lymphomas in man;    -   4/ human T cell leukemia virus HTLV-I and HTLV-II receptors,        recently mapped to chromosome 17 (Sommerfelt M. A. et al.,        Science 242 (1988) 1557-1559), these viruses being responsible        for adult T cell leukemia as well as for tropical spastic        paraparesie (HTLV-I) and tricholeucocytic leukemia (HTLV-II);    -   5/ the receptor of the ecotropic murine leukemia virus MuLV-E,        mapped to chromosome 5 of the mouse by Oie et al. (Nature        274 (1978) 60-62) and recently cloned by Albritton et al. (Cell        57 (1989) 659-666).

Another goal of the present invention concerns the development of stablehybrid macromolecules with an anticancer function, obtained by thecoupling of albumin variants with molecules able to bind growth factorswhich, in certain pathologies associated with the amplification of thecorresponding membraneous proto-oncogenes, can interact with theirtarget cells and induce a transformed phenotype. An example of suchreceptors is the class of receptors with tyrosine kinase activity(Yarden Y. and Ulrich A., Biochemistry 27 (1988) 3113-3119), the bestknown being the epidermal growth factor (EGF) and the colony stimulatingfactor I (CSF-I) receptors, respectively coded by the proto-oncogenesc-erbB-1 (Downward J. et al., Nature 307 (1984) 521-527) and c-fms(Sherr C. J. et al., Cell 41 (1985) 665-676). Another example of suchreceptors includes the human insulin receptor (HIR), theplatelet-derived growth factor (PDGF) receptor, the insulin-like growthfactor I (IGF-I) receptor, and most notably the proto-oncogene c-erbB-2,whose genomic amplification and/or overexpression was shown to bestrictly correlated with certain human cancers, in particular breastcancer (Slamon D. J. et al., Science 235 (1987) 177-182; Kraus M. H. etal., EMBO J. 6 (1987) 605-610). Furthermore, the principles put forth inthe present invention can be equally applied to other receptors, forexample the interleukin 6 (IL-6) receptor, which has been shown in vitroto be an autocrine factor in renal carcinoma cells (Miki S. et al., FEBSLett., 250 (1989) 607-610).

As indicated above, the hybrid macromolecules of interest aresubstantially preferably proteinic and can therefore be generated by thetechniques of genetic engineering. The preferred way to obtain thesemacromolecules is by the culture of cells transformed, transfected, orinfected by vectors expressing the macromolecule. In particular,expression vectors capable of transforming yeasts, especially of thegenus Kluyveromyces, for the secretion of proteins will be used. Such asystem allows for the production of high quantities of the hybridmacromolecule in a mature form, which is secreted into the culturemedium, thus facilitating purification.

The preferred method for expression and secretion of the hybridmacromolecules consists therefore of the transformation of yeast of thegenus Kluyveromyces by expression vectors derived from theextrachromosomal replicon pKD1, initially isolated from K. marxianusvar. drosophilarum. These yeasts, and in particular K. marxianus(including the varieties lactis, drosophilarum and marxianus which arehenceforth designated respectively as K. lactis, K. drosophilarum and K.fragilis), are generally capable of replicating these vectors in astable fashion and possess the further advantage of being included inthe list of G.R.A.S. (“Generally Recognized As Safe”) organisms. Theyeasts of particular interest include industrial strains ofKluyveromyces capable of stable replication of said plasmid derived fromplasmid pKD1 into which has been inserted a selectable marker as well asan expression cassette permitting the secretion of the given hybridmacromolecule at high levels.

Three types of cloning vectors have been described for Kluyveromyces:

-   -   i) Integrating vectors containing sequences homologous to        regions of the Kluyveromyces genome and which, after being        introduced into the cells, are integrated in the Kluyveromyces        chromosomes by in vivo recombination (International patent        application WO 83/04050). Integration, a rare event requiring an        efficient selection marker, is obtained when these vectors do        not contain sequences permitting autonomous replication in the        cell. The advantage of this system is the stability of the        transformed strains, meaning that they can be grown in a normal        nutritive medium without the need for selection pressure to        maintain the integrated sequences. The disadvantage, however, is        that the integrated genes are present in only a very small        number of copies per cell, which frequently results in a low        level of production of a heterologous protein.    -   ii) Replicating vectors containing Autonomously Replicating        Sequences (ARS) derived from the chromosomal DNA of        Kluyveromyces (Das S. and Hollenberg C. P., Current Genetics        6 (1982) 123-128; International patent application WO 83/04050).        However these vectors are of only moderate interest, since their        segregation in mitotic cell division is not homogeneous, which        results in their loss from the cells at high frequency even        under selection pressure.    -   iii) Replicating vectors derived from naturally occurring yeast        plasmids, either from the linear “killer” plasmid k1 isolated        from K. lactis (de Louvencourt L. et al., J. Bacteriol.        154 (1983) 737-742; European patent application EP 0 095 986 A1,        publ. Dec. 7, 1983), or from the circular plasmid pKD1 isolated        from K. drosophilarum (Chen X. J. et al., Nucl. Acids Res.        14 (1986) 4471-4480; Falcone C. et al., Plasmid 15 (1986)        248-252; European patent application EP 0 241 435 A2, publ. Oct.        14, 1987). The vectors containing replicons derived from the        linear “killer” plasmid require a special nutrient medium, and        are lost in 40-99% of the cells after only 15 generations, even        under selection pressure (European patent application EP 0 095        986 A1, 1983), which limits their use for mass production of        heterologous proteins.

The vectors derived from plasmid pKD1 described in European patentapplication EP 0 241 435 A2 are also very unstable since even the mostperformant vector (P3) is lost in approximately 70% of the cells afteronly six generations under nonselective growth conditions.

An object of the present invention concerns the utilization of certainplasmid constructions derived from the entire pKD1 plasmid; suchconstructions possess significantly higher stability characteristicsthan those mentioned in European patent application EP 0 241 435 A2. Itwill be shown in the present invention that these new vectors are stablymaintained in over 80% of the cells after 50 generations undernonselective growth conditions.

The high stability of the vectors used in the present invention wasobtained by exploiting fully the characteristics of plasmid pKD1.Besides an origin of replication, this extrachromosomal replicon systempossesses two inverted repeats, each 346 nucleotides in length, andthree open reading frames coding for genes A, B et C, whose expressionis crucial for plasmid stability and high copy number. By analogy withthe 2μ plasmid of S. cerevisiae, which is structurally related toplasmid pKD1 (Chen X. J. et al., Nucl. Acids Res. 14 (1986) 4471-4480),the proteins encoded by genes B et C are probably involved in plasmidpartitioning during mitotic cell division, and may play a role in thenegative regulation of gene A which encodes a site-specific recombinase(FLP). It has been shown that the FLP-mediated recombination between theinverted repeats of the 2μ plasmid of S. cerevisiae is the basis of amechanism of autoregulation of the number of plasmid copies per cell:when copy number becomes too low to permit the production of sufficientquantities of the products of genes B and C, which act as repressors ofgene A, the FLP recombinase is induced and the plasmid replicatesaccording to a rolling circle type model, which amplifies copy number toabout 50 copies per cell (Futcher A. B., Yeast 4 (1988) 2740).

The vectors published in European patent application EP 0 241 435 A2 donot possess the above-mentioned structural characteristics of plasmidpKD1 of K. drosophilarum: vector A15 does not carry the completesequence of pKD1, and vectors P1 and P3 carry an interrupted A gene,thereby destroying the system of autoregulated replication of residentplasmid pKD1. In contrast, the pKD1-derived constructs used in thepresent invention maintain the structural integrity of the invertedrepeats and the open reading frames A, B and C, resulting in a notablyhigher stability of the plasmid as well as an increased level ofsecretion of the therapeutically active hybrid macromolecules.

The expression cassette will include a transcription initiation region(promoter) which controls the expression of the gene coding for thehybrid macromolecule. The choice of promoters varies according to theparticular host used. These promoters derive from genes of Saccharomycesor Kluyveromyces type yeasts, such as the genes encodingphosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase(GPD), the lactase of Kluyveromyces (LAC4), the enolases (ENO), thealcohol dehydrogenases (ADH), the acid phosphatase of S. cerevisiae(PHO5), etc . . . These control regions may be modified, for example byin vitro site-directed mutagenesis, by introduction of additionalcontrol elements or synthetic sequences, or by deletions orsubstitutions of the original control elements. For example,transcription-regulating elements, the so-called “enhancers” of highereukaryotes and the “upstream activating sequences” (UAS) of yeasts,originating from other yeast promoters such as the GAL1 and GAL10promoters of S. cerevisiae or the LAC4 promoter of K. lactis, or eventhe enhancers of genes recognized by viral transactivators such as theE2 transactivator of papillomavirus, can be used to construct hybridpromoters which enable the growth phase of a yeast culture to beseparated from the phase of expression of the gene encoding the hybridmacromolecule. The expression cassette used in the present inventionalso includes a transcription and translation termination region whichis functional in the intended host and which is positioned at the 3′ endof the sequence coding for the hybrid macromolecule.

The sequence coding for the hybrid macromolecule will be preceded by asignal sequence which serves to direct the proteins into the secretorypathway. This signal sequence can derive from the natural N-terminalregion of albumin (the prepro region), or it can be obtained from yeastgenes coding for secreted proteins, such as the sexual pheremones or thekiller toxins, or it can derive from any sequence known to increase thesecretion of the so-called proteins of pharmaceutical interest,including synthetic sequences and all combinations between a “pre” and a“pro” region.

The junction between the signal sequence and the sequence coding for thehybrid macromolecule to be secreted in mature form corresponds to a siteof cleavage of a yeast endoprotease, for example a pair of basic aminoacids of the type Lys⁻²-Arg⁻¹ or Arg⁻²-Arg⁻¹ corresponding to therecognition site of the protease coded by the KEX2 gene of S. cerevisiaeor the KEX1 gene of K. lactis (Chen X. J. et al., J. Basic Microbiol. 28(1988) 211-220; Wesolowski-Louvel M. et al., Yeast 4 (1988) 71-81). Infact, the product of the KEX2 gene of S. cerevisiae cleaves the normal“pro” sequence of albumin in vitro but does not cleave the sequencecorresponding to the pro-albumin “Christchurch” in which the pair ofbasic amino acids is mutated to Arg⁻²-Glu⁻¹ (Bathurst I. C. et al.,Science 235 (1987) 348-350).

In addition to the expression cassette, the vector will include one orseveral markers enabling the transformed host to be selected. Suchmarkers include the URA3 gene of yeast, or markers conferring resistanceto antibiotics such as geneticin (G418), or any other toxic compoundsuch as certain metal ions. These resistance genes will be placed underthe control of the appropriate transcription and translation signalsallowing for their expression in a given host.

The assembly consisting of the expression cassette and the selectablemarker can be used either to directly transform yeast, or can beinserted into an extrachromosomal replicative vector. In the first case,sequences homologous to regions present on the host chromosomes will bepreferably fused to the assembly. These sequences will be positioned oneach side of the expression cassette and the selectable marker in orderto augment the frequency of integration of the assembly into the hostchromosome by in vivo recombination. In the case where the expressioncassette is inserted into a replicative vector, the preferredreplication system for Kluyveromyces is derived from the plasmid pKD1initially isolated from K. drosophilarum, while the preferredreplication system for Saccharomyces is derived from the 2μ plasmid. Theexpression vector can contain all or part of the above replicationsystems or can combine elements derived from plasmid pKD1 as well as the2μ plasmid.

When expression in yeasts of the genus Kluyveromyces is desired, thepreferred constructions are those which contain the entire sequence ofplasmid pKD1. Specifically, preferred constructions are those where thesite of insertion of foreign sequences into pKD1 is localized in a 197bp region lying between the SacI (SstI) site and the MstII site, oralternatively at the SphI site of this plasmid, which permits highstability of the replication systems in the host cells.

The expression plasmids can also take the form of shuttle vectorsbetween a bacterial host such as Escherichia coli and yeasts; in thiscase an origin of replication and a selectable marker that function inthe bacterial host would be required. It is also possible to positionrestriction sites which are unique on the expression vector such thatthey flank the bacterial sequences. This allows the bacterial sequencesto be eliminated by restriction cleavage, and the vector to be religatedprior to transformation of yeast, and this can result in a higherplasmid copy number and enhanced plasmid stability. Certain restrictionsites such as 5′-GGCCNNNNNGGCC-3′ (SfiI) or 5′-GCGGCCGC-3′ (NotI) areparticularly convenient since they are very rare in yeasts and aregenerally absent from an expression plasmid.

The expression vectors constructed as described above are introducedinto yeasts according to classical techniques described in theliterature. After selection of transformed cells, those cells expressingthe hybrid macromolecule of interest are inoculated into an appropriateselective medium and then tested for their capacity to secrete the givenprotein into the extracellular medium. The harvesting of the protein canbe conducted during cell growth for continuous cultures, or at the endof the growth phase for batch cultures. The hybrid proteins which arethe subject of the present invention are then purified from the culturesupernatant by methods which take into account their molecularcharacteristics and pharmacological activities.

The present invention also concerns the therapeutic application of thehybrid macromolecules described therein, notably in the treatment andthe prevention of AIDS, as well as the cells which are transformed,transfected, or infected by vectors expressing such macromolecules.

The examples which follow as well as the attached figures show some ofthe characteristics and advantages of the present invention.

DESCRIPTION OF FIGURES

The diagrams of the plasmids shown in the figures are not drawn toscale, and only the restriction sites important for the constructionsare indicated.

FIG. 1: Oligodeoxynucleotides used to generate the MstII andHindIII-SmaI restriction sites, situated respectively upstream anddownstream of the V1V2 domains of the CD4 molecule.

FIG. 2: Nucleotide sequence of the MstII-SmaI restriction fragmentincluding the V1 and V2 domains of the CD4 receptor of the HIV-1 virus.The recognition sites for MstII, HindIII and SmaI are underlined.

FIG. 3: Construction of plasmid pXL869 coding for prepro-HSA.

FIG. 4: Construction of plasmids pYG208 and pYG210.

FIG. 5: Construction of plasmid pYG11.

FIG. 6: Construction of plasmid pYG18.

FIG. 7: Restriction map of plasmid pYG303.

FIG. 8: Nucleotide sequence of restriction fragment HindIII coding forthe protein fusion prepro-HSA-V1V2. Black arrows indicate the end of the“pre” and “pro” regions of HSA. The MstII site is underlined.

FIG. 9: Restriction map of plasmid pYG306.

FIG. 10: Construction of plasmid pUC-URA3.

FIG. 11: Construction of plasmid PCXJ1.

FIG. 12: Construction of plasmid pk1-PS1535-6.

FIG. 13: Construction of plasmids pUC-kan1 and pUC-kan202.

FIG. 14: Construction of plasmid pKan707.

FIG. 15: Stability curve of plasmid pKan707 in strain MW98-8C undernonselective growth conditions.

FIG. 16: Construction of plasmid pYG308B.

FIG. 17: Construction of plasmid pYG221B.

FIG. 18: Characterization of the material secreted after 4 days inculture by strain MW98-8C transformed by plasmids pYG221B (prepro-HSA)and pYG308B (prepro-HSA-V1V2). A, Coomassie staining afterelectrophoretic migration in an 8.5% polyacrylamide gel. Molecularweight standards (lane 1); supernatant equivalent to 300 μl of theculture transformed by plasmid pYG308B (lane 2); supernatant equivalentto 100 μl of the culture transformed by plasmid pYG221B (lane 3); 500 ngof HSA (lane 4). B, immunologic characterization of the secretedmaterial subject to electrophoretic migration in an 8.5% polyacrylamidegel, followed by transfer to a nitrocellulose membrane and utilizationof primary antibodies directed against human albumin: 250 ng of HSAstandard (lane 1); supernatant equivalent to 100 μl of the culturetransformed by plasmid pYG308B (lane 2); supernatant equivalent to 10 μlof the culture transformed by plasmid pYG221B (lane 3). C, exactly as inB except that polyclonal antibodies directed against the CD4 moleculewere used in place of antibodies directed against HSA.

FIG. 19: Titration of the protein HSA-V1V2 (1 μg/ml) by mouse monoclonalantibody Leu3A (Becton Dickinson, Mountain View, Calif., U.S.A.) (panelA), by mouse monoclonal antibody OKT4A (Ortho Diagnostic Systems,Raritan, N.J., USA) (panel B), or by polyclonal goat anti-HSA coupled toperoxidase (Nordic, Tilburg, Netherlands) (panel C). After usingantibodies Leu3A and OKT4A, a secondary rabbit anti-mouse antibodycoupled to peroxidase (Nordic) is used. Titration curves for the threeprimary antibodies used in parts A, B and C were determined by measuringoptical density at 405 nm after addition of a chromogenic substrate ofperoxidase (ABTS, Fluka, Switzerland). Ordinate: OD at 405 nm, abscissa:dilution factor of the primary antibody used.

FIG. 20: Assay of protein HSA-V1V2 by the ELISA sandwich method: rabbitpolyclonal anti-HSA (Sigma)/HSA-V1V2/mouse monoclonal antibody Leu3A(Becton Dickinson) (panel A), or rabbit polyclonal anti-HSA(Sigma)/HSA-V1V2/mouse monoclonal antibody OKT4A (Ortho DiagnosticSystems) (panel B). After incubation of each antibody with the HSA-V1V2protein, a secondary rabbit anti-mouse antibody coupled to peroxidase(Nordic) is added. Titration curves were determined by measuring opticaldensity at 405 nm after addition of the peroxidase substrate ABTS.Ordinate: OD at 405 nm; abscissa: concentration of HSA-V1V2 in μg/ml.

FIG. 21: Soluble phase inhibition of binding to CD4 by 125 femtomoles ofrecombinant gp160 protein (Transgene, Strasbourg, France). Opticaldensity at 492 nm is represented on the ordinate (the value 2 is thesaturation optical density of the system) and the quantities of HSA(control), HSA-CD4, and soluble CD4 are shown on the abscissa (picomolesof protein).

FIG. 22: Inhibition of the binding of inactivated HIV-1 virus to cellline CEM13. A, preliminary analysis of cell populations sorted as afunction of their fluorescence. Ordinate: cell number; abscissa:fluorescence intensity (logarithmic scale). B, histogram of cellpopulations sorted as a function of their fluorescence. Column 1,negative control; Column 2, HIV-1 virus; Column 3, HIV-1 viruspreincubated with 116 picomoles of CD4 recombinant protein; Column 4,HIV-1 virus preincubated with 116 picomoles of HSA-V1V2; Column 5, HIV-1virus preincubated with 116 picomoles of HSA.

FIG. 23: Inhibition of infection in cell culture. Reverse transcriptaseactivity was measured for 19 days after infection of CEM13 cells. Assayswere performed on microtitration plates according to the followingprotocol: into each well, 10 μl of Buffer A (0.5 M KCl, 50 mM DTT, 0.5%Triton X-100), then 40 μl of Buffer B (10 μl 5 mM EDTA in 0.5 M Tris-HClpH 7.8, 1 μl 0.5 M MgCl₂, 3 μl ³H-dTTP, 10 μl poly rA-oligodT at 5OD/ml, 16 μl H₂O) were added to 50 μl culture supernatant removed atdifferent times after infection. The plates were incubated for 1 hour at37° C., then 20 μl of Buffer C (120 mM Na₄P₂O₇ in 60% TCA) was added andincubation was continued for 15 minutes at 4° C. The precipitates formedwere passed through Skatron filters using a Skatron cell harvester, andwashed with Buffer D (12 mM Na₄P₂O₇ in 5% TCA). Filters were dried 15minutes at 80° C. and the radioactivity was measured in a scintillationcounter. Three independent samples were tested for each point.

FIG. 24: Changes in the in vivo concentrations of CD4, HSA and HSA-CD4over time.

FIG. 25: Construction of plasmids pYG232, pYG233 and pYG364.

FIG. 26: Construction of plasmid pYG234.

FIG. 27: Construction of plasmids pYG332 and pYG347.

FIG. 28: Construction of plasmids pYG362, pYG363 and pYG511.

FIG. 29: Restriction maps of plasmids pYG371, pYG374 and pYG375.

FIG. 30: Restriction map of expression plasmid pYG373B.

FIG. 31: Construction of plasmid pYG537.

FIG. 32: Construction of expression plasmid pYG560.

FIG. 33: Intracellular expression of hybrid proteins HSA-V1 (plasmidpYG366B; lane b), V1-HSA (plasmid pYG373B; lane c), V1-HSA-V1V2 (plasmidpYG380B; lane d), V1-HSA-V1 (plasmid pYG381B, lane e) and HSA-V1V2(plasmid pYG308B, lane f) in K. lactis strain MW98-8C. Detection wasperformed by the Western Blot method using polyclonal rabbit serumdirected against HSA as primary antibody. 10 μg of protein from theinsoluble fraction was loaded in each case.

FIG. 34: Introduction of the “Leucine Zipper” of c-jun (BglII-AhaIIfragment) in a hybrid protein HSA-CD4.

FIG. 35: Secretion in strain MW98-8C of truncated HSA variants coupledto the V1V2 domains of the CD4 receptor. Panel 1: Coomassie bluestaining. Each lane was loaded with the equivalent of 400 μl of culturesupernatant from the early stationary phase. Molecular weight markers(lane a), strain transformed by control vector pKan707 (lane b), HSAstandard (lane c), strain transformed by expression plasmids pYG308B(HSA₅₈₅-V₁V2, lane d), pYG334B (HSA₃₁₂-V1V2, lane e), and pYG335B(HSA₃₀₀-V1V2, lane f).

-   -   Panel 2: Western Blot detection using rabbit polyclonal        anti-HSA. Each lane was loaded with the equivalent of 100 μl of        culture supernatant from the early stationary phase.        Biotinylated molecular weight markers (Bio-Rad, lane a), strain        transformed by control vector pKan707 (lane b), HSA standard        (lane f), strain transformed by expression plasmids pYG308B        (HSA₅₈₅-V1V2, lane c), pYG334B (HSA₃₁₂-V1V2, lane d), and        pYG335B (HSA₃₀₀-V1V2, lane e). Panel 3: Western Blot detection        using a rabbit polyclonal anti-CD4 serum; same legend as in        Panel 2.

FIG. 36: Panel a: representation of several HindIII(−25)-MstIIrestriction fragments corresponding to deletions in HSA. Amino acidposition (numbered according to mature HSA) is indicated in parentheses.Panel b: detail of the position of the MstII site in one of thedeletants (clone YP63, linker insertion at amino acid 495).

FIG. 37: Examples of the hinge regions between the HSA and CD4 moieties.The amino acid pairs that are potential targets of endoproteasesinvolved in the secretory pathway are boxed. Panel 1: hinge region ofprotein HSA₅₈₅-CD4. Panel 2: hinge region of HSA_(Bal31)-CD4 proteinsobtained by Bal31 deletion of the C-terminal portion of HSA (in thisrepresentation the Lys-Lys pairs situated at the beginning of the CD4moiety have been modified by site-directed mutagenesis as exemplified inE.13.2.). Panel 3: hinge region obtained by insertion of a polypeptide(shown here a fragment of troponin C), obtained after site-directedmutagenesis using oligodeoxynucleotide Sq1445. Panel 4: generalstructure of the hinge region between the HSA and CD4 moieties.

FIG. 38: Panel 1: structure of the in-frame fusion between the preproregion of HSA and the CD4 receptor, present notably in expressionplasmids pYG373B, pYG380B, pYG381B and pYG560. Panel 1a: the amino acidpairs that are potential targets of endoproteases involved in thesecretory pathway are boxed. Panel 1b: These amino acid pairs can bemodified by mutating the second lysine of each pair such that the pairis no longer a target for such endoproteases. Panel 2: Examples of hingeregions between the CD4 and HSA moieties present notably in hybridproteins V1-HSA (panel 2a) or V1V2-HSA (panels 2b and 2c). Panel 3:general structure of the hinge region between the CD4 and HSA moieties.

EXAMPLES

General Cloning Techniques.

The classical methods of molecular biology such as preparativeextractions of plasmid DNA, the centrifugation of plasmid DNA in cesiumchloride gradients, agarose and polyacrylamide gel electrophoresis, thepurification of DNA fragments by electroelution, the extraction ofproteins by phenol or phenol/chloroform, the precipitation of DNA in thepresence of salt by ethanol or isopropanol, transformation ofEscherichia coli etc . . . have been abundantly described in theliterature (Maniatis T. et al., “Molecular Cloning, a LaboratoryManual”, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982;Ausubel F. M. et al. (eds), “Current Protocols in Molecular Biology”,John Wiley & Sons, New York, 1987), and will not be reiterated here.

Restriction enzymes are furnished by New England Biolabs (Biolabs),Bethesda Research Laboratories (BRL) or Amersham and are used accordingto the recommendations of the manufacturer.

Plasmids pBR322, pUC8, pUC19 and the phages M13 mp8 and M13mp18 are ofcommercial origin (Bethesda Research Laboratories).

For ligations, the DNA fragments are separated by size on agarose(generally 0.8%) or polyacrylamide (generally 10%) gels, purified byelectroelution, extracted with phenol or phenol/chloroform, precipitatedwith ethanol and then incubated in the presence of T4 DNA ligase(Biolabs) according to the recommendations of the manufacturer.

Filling in of 5′ ends is carried out using the Klenow fragment of E.coli DNA polymerase I (Biolabs) according to manufacturerrecommendations. Destruction of 3′ protruding termini is performed inthe presence of T4 DNA polymerase (Biolabs) as recommended by themanufacturer. Digestion of 5′ protruding ends is accomplished by limitedtreatment with S1 nuclease.

In vitro site-directed mutagenesis is performed according to the methoddeveloped by Taylor et al. (Nucleic Acids Res. 13 (1985) 8749-8764)using the kit distributed by Amersham.

Enzymatic amplification of DNA fragments by the PCR technique(Polymerase-catalyzed Chain Reaction, Saiki R. K. et al., Science 230(1985) 1350-1354; Mullis K. B. and Faloona F. A., Meth. Enzym. 155(1987) 335-350) is carried out on a “DNA thermal cycler” (Perkin ElmerCetus) according to manufacturer specifications.

Nucleotide sequencing is performed according to the method developed bySanger et al. (Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467), usingthe Amersham kit.

Transformation of K. lactis with foreign DNA as well as the purificationof plasmid DNA from K. lactis are described in the text.

Unless indicated otherwise, the bacterial strains used are E. coliMC1060 (lacIPOZYA, X74, galU, galK, strA^(r)), or E. coli TG1 (lac proA,B, supE, thi, hsdD5/F′traD36, proA⁺B⁺, lacI^(q), lacZ, M15).

All yeast strains used are members of the family of budding yeasts andin particular of the genus Kluyveromyces. Examples of these yeasts aregiven in the text. The K. lactis strain MW98-8C (α, uraA arg, lys, K⁺,pKD1°) was often used; a sample of this strain has been deposited onSep. 16, 1988 at the Centraalbureau voor Schimmelkulturen (CBS) at Baarn(Netherlands) under the registration number CBS 579.88.

Example 1 Construction of a MstII/HindIII-SmaI Restriction FragmentCarrying the V1V2 Domains of the Receptor of the HIV-1 Virus

An MstII-SmaI restriction fragment corresponding to the V1V2 domains(where V1 and V2 designate the first two N-terminal domains of the CD4molecule) was generated by the technique of enzymatic amplification(PCR) according to the following strategy: the lymphoblastic cell lineCEM13, which expresses high quantities of CD4 receptor, was used as thesource of messenger RNAs coding for the receptor. Total RNA was firstpurified from 3×10⁸ cells of this line by extraction with guanidiumthiocyanate as originally described by Cathala et al. (DNA 4 (1983)329-335); 50 μg of RNA prepared in this manner then served as matrix forthe synthesis of complementary DNA (cDNA) using the Amersham kit and theoligodeoxynucleotide Xol27 as primer (FIG. 1). The resulting cDNA wassubjected to 30 cycles of enzymatic amplification by the PCR techniqueat a hybridization temperature of 62° C., using 1 μg each ofoligodeoxynucleotides Xol26 and Xol27 as primer, as shown in FIG. 1. Theamplified fragment was directly cloned into the SmaI site of M13mp8which had been previously dephosphorylated, to generate vector M13/CD4.This vector is an intermediate construction containing the restrictionfragment MstII-SmaI which itself is the source of the MstII-HindIIIfragment carrying the V1V2 domains of the CD4 molecule; the nucleotidesequence of this fragment is shown in FIG. 2.

Example 2 Construction of the Expression Cassette for Prepro-HSA

E.2.1. Construction of Plasmid pXL869 Coding for Prepro-HSA.

The NdeI site of plasmid pXL322 (Latta M. et al., Bio/Technology 5(1987) 1309-1314) including the ATG translation initiation codon ofprepro-HSA was changed to a HindIII site byoligodeoxynucleotide-directed mutagenesis using the following strategy:the HindIII-BglII fragment of pXL322 containing the 5′ extremity of theprepro-HSA gene was cloned into vector M13mp18 and mutagenized witholigodeoxynucleotide 5′-ATCTAAGGAAATACAAGCTT-ATGAAGTGGGT-3′ (the HindIIIsite is underlined and the ATG codon of prepro-HSA is shown in boldtype); the phage obtained after this mutagenesis step is plasmid pXL855whose restriction map is shown in FIG. 3. After verification of thenucleotide sequence, the complete coding sequence for prepro-HSA wasreconstituted by ligation of the HindIII-PvuII fragment derived from thereplicative form of the mutagenized phage and coding for the N-terminalregion of prepro-HSA, with the PvuII-HindIII fragment of plasmid pXL322containing the C-terminal of HSA, thereby generating a HindIII fragmentcoding the entire prepro-HSA gene. This HindIII fragment, which alsocontains a 61 bp nontranslated region at its 3′ extremity, was clonedinto the corresponding site of plasmid pUC8 to generate plasmid pXLS69(FIG. 3).

E2.2. Construction of Expression Cassettes for Prepro-HSA ExpressedUnder the Control of the PGK Promoter of S. cerevisiae.

Plasmid pYG12 contains a 1.9 kb SalI-BamHI restriction fragment carryingthe promoter region (1.5 kb) and terminator region (0.4 kb) of the PGKgene of S. cerevisiae (FIG. 4). This fragment is derived from a genomicHindIII fragment (Mellor J. et al., Gene 24 (1983) 1-14) from which a1.2 kb fragment corresponding to the structural gene has been deleted,comprising a region between the ATG translation initiation codon and theBglII site situated 30 codons upstream of the TAA translationtermination codon. The HindIII sites flanking the 1.9 kb fragment werethen destroyed using synthetic oligodeoxynucleotides and replaced by aSalI and a BamHI site respectively upstream of the promoter region anddownstream of the transcription terminator of the PGK gene. A uniqueHindIII site was then introduced by site-directed mutagenesis at thejunction of the promoter and terminator regions; the sequence flankingthis unique HindIII site (shown in bold letters) is as follows:5′-TAAAAACAAAAGATCCCCAAGCTTGGGGATCTCCCATGTCTC TACT-3′

Plasmid pYG208 is an intermediate construction generated by insertion ofthe synthetic adaptor BamHI/SalI/BamHI (5′-GATCCGTCGACG-3′) into theunique BamHI site of plasmid pYG12; plasmid pYG208 thereby allows theremoval of the promoter and terminator of the PGK gene of S. cerevisiaein the form of a SalI restriction fragment (FIG. 4).

The HindIII fragment coding for prepro-HSA was purified from plasmidpXL869 by electroelution and cloned in the “proper” orientation (definedas the orientation which places the N-terminal of the albumin preproregion just downstream of the PGK promoter) into the HindIII site ofplasmid pYG208 to generate plasmid pYG210. As indicated in FIG. 4,plasmid pYG210 is the source of a SalI restriction fragment carrying theexpression cassette (PGK promoter/prepro-HSA/PGK terminator).

E.2.3. Optimization of the Expression Cassette.

The nucleotide sequence located immediately upstream of the ATGtranslation initiation codon of highly expressed genes possessesstructural characteristics compatible with such high levels ofexpression (Kozak M., Microbiol. Rev. 47 (1983) 1-45; Hamilton R et al.,Nucl. Acid Res. 15 (1987) 3581-3593). The introduction of a HindIII siteby site-directed mutagenesis at position-25 (relative to the ATGinitiation codon) of the PGK promoter of S. cerevisiae is described inEuropean patent application EP No 89 10480.

In addition, the utilization of oligodeoxynucleotides Sq451 and Sq452which form a HindIII-BstEII adaptor is described in the same documentand permits the generation of a HindIII restriction fragment composed ofthe 21 nucleotides preceding the ATG initiator codon of the PGK gene,followed by the gene coding for prepro-HSA. The nucleotide sequencepreceding the ATG codon of such an expression cassette is as follows(the nucleotide sequence present in the PGK promoter of S. cerevisiae isunderlined): 5′-AAGCTTTACAACAAATATAAAAACAATG-3′.

Example 3 In-Frame Fusion of Prepro-HSA with the V1V2 Domains of the CD4Receptor

The cloning strategy used for the in-frame construction of the hybridmolecule prepro-HSA-V1V2 is illustrated in FIGS. 5 through 9. PlasmidpYG11 is an intermediate construction in which the HindIII fragmentcoding for prepro-HSA has been purified from plasmid pXL869 and clonedinto the HindIII site of plasmid pYG12 (FIG. 5). The construction ofplasmid pYG18 is represented in FIG. 6; this plasmid corresponds to theSalI-BamHI fragment coding for the expression cassette (PGKpromoter/prepro-HSA/PGK terminator) purified from plasmid pYG11 andcloned into the corresponding sites of plasmid pIC20R (Marsh F. et al.,Gene 32 (1984) 481-485).

The MstII-SmaI restriction fragment carrying the V1V2 domains of the CD4receptor, obtained as described in Example 1, was cloned into plasmidpYG18 cut by the same enzymes to generate recombinant plasmid pYG303whose restriction map is shown in FIG. 7. Plasmid pYG303 thereforecarries a HindIII fragment corresponding to the in-frame fusion of theentire prepro-HSA gene followed by the V1V2 domains of the CD4 receptor;FIG. 8 shows the nucleotide sequence of this fragment. This fragment wasthen cloned into the HindIII site of plasmid pYG208: insertion of thisfragment, which codes for the gene prepro-HSA-V1V2, in the properorientation into plasmid pYG208, generates plasmid pYG306 whoserestriction map is shown in FIG. 9. Plasmid pYG306 carries a SalIrestriction fragment containing the expression cassette (PGKpromoter/prepro-HSA-V1V2/PGK terminator).

Example 4 Construction of Stable Cloning Vectors Derived from RepliconpKD1

E4.1. Isolation and Purification of Plasmid pKD1.

Plasmid pKD1 was purified from K. drosophilarum strain UCD 51-130(U.C.D. collection, University of California, Davis, Calif. 95616)according to the following protocol: a 1 liter culture in YPD medium (1%yeast extract, 2% Bacto-peptone, 2% glucose) was centrifuged, washed,and resuspended in a solution of 1.2 M sorbitol, and cells weretransformed into spheroplasts in the presence of zymolyase (300 μg/ml),25 mM EDTA, 50 mM phosphate and β-mercaptoethanol (1 μg/ml). Afterwashing in a solution of 1.2 M sorbitol, spheroplasts corresponding to250 ml of the original culture were resuspended in 2.5 ml of 1.2 Msorbitol to which was added the same volume of buffer (25 mM Tris-HCl,pH 8.0; 50 mM glucose; 10 mM EDTA). The following steps correspond tothe alkaline lysis protocol already described (Birnboim H. C. and DolyJ. C., Nucleic Acids Res. 7 (1979) 1513-1523). DNA was purified byisopycnic centrifugation in a cesium chloride gradient.

E4.1 Construction of Plasmid pCXJ1.

The intermediate construction pUC-URA3 (FIG. 10) consists of a 1.1 kbfragment containing the URA3 gene of S. cerevisiae inserted in theunique NarI site of plasmid pUC19 as follows: the HindIII fragmentcoding for the URA3 gene was purified by HindIII digestion of plasmidpG63 (Gerbaud C. et al., Curr. Genet. 3 (1981) 173-180); the fragmentwas treated with the Klenow fragment of E. coli DNA polymerase I togenerate blunt ends, purified by electroelution, and inserted intoplasmid pUC19 which had been cleaved by NarI and treated with the Klenowfragment of E. coli DNA polymerase I.

Plasmid pCXJ1 (FIG. 11) contains the complete sequence of plasmid pKD1inserted into the unique AatII site of pUC-URA3 as follows: plasmid pKD1was linearized by cleavage with EcoRI, then blunt-ended with the Klenowfragment of E. coli DNA polymerase I. This fragment was then ligatedwith plasmid pUC-URA3 which had been cut by AatII and blunt-ended withT4 DNA polymerase: cloning of a blunt-ended EcoRI fragment into ablunt-ended AatII site reconstitutes two EcoRI sites. It should be notedthat linearization of plasmid pKD1 at the EcoRI site does not inactivateany of the genes necessary for plasmid stability and copy number, sincethe EcoRI site is located outside of genes A, B, and C, and outside ofthe inverted repeats of pKD1. In fact, plasmid pCXJ1 transforms K.lactis uraA cir° at high frequency, is amplified to 70-100 copies percell, and is maintained in a stable fashion in the absence of selectionpressure. Due to the origin of replication carried by plasmid pUC-URA3,plasmid PCXJ1 can also replicate in E. coli and thus constitutes aparticularly useful shuttle vector between E. coli and several yeasts ofthe genus Kluyveromyces, in particular K. lactis, K. fragilis and K.drosophilarum. However, the utilization of pCXJ1 as a vector for thetransformation of Kluyveromyces remains limited to those auxotrophicstrains carrying a chromosomal uraA mutation.

E.4.3. Construction of an In-Frame Fusion Between ORF1 of the KillerPlasmid of K. lactis and the Product of the Bacterial Gene aph[3′]-I ofTransposon Tn903.

Plasmid pKan707 was constructed as a vector to be used in wild typeyeasts. This plasmid was generated by insertion of the aph[3′]-I gene ofbacterial transposon Tn903 coding for 3′-aminoglycosidephosphotransferase (APH), expressed under control of a yeast promoter,into the SaI of plasmid PCXJ1.

In the first step, the bacterial transcription signals of the aph[3′]-Igene were replaced by the P_(k1) promoter isolated from the killerplasmid k1 of K. lactis as follows: the 1.5 kb ScaI-PstI fragment ofplasmid k1 was cloned into the corresponding sites of vector pBR322, togenerate plasmid pk1-PS1535-6 (FIG. 12); this 1.5 kb fragment containsthe 5′ region of the first open reading frame (ORF1) carried by plasmidk1 as well as approximately 220 bp upstream (Sor F. and Fukuhara H.,Curr. Genet. 9 (1985) 147-155). The purified ScaI-PstI fragment probablycontains the entire promoter region of ORF1, since the ScaI site issituated only 22 nucleotides from the extremity of plasmid k1 (Sor F.and al., Nucl. Acids. Res. 11 (1983) 5037-5044). Digestion ofpk1-PS1535-6 by DdeI generates a 266 bp fragment containing 17 bp frompBR322 at the extremity dose to the ScaI site, and the first 11 codonsof ORF1 at the other extremity.

Plasmid pUC-kan1 is an intermediate construction obtained by insertionof the 1.25 kb EcoRI fragment carrying the aph[3′]-I gene of Tn903(Kanamycin Resistance Gene Block™, Pharmacia), into the EcoRI site ofplasmid pUC19 (FIG. 13). The 266 bp DdeI fragment from plasmidpk1-PS1535-6 was treated with the Klenow fragment of E. coli DNApolymerase I, purified by electroelution on a polyacrylamide gel, theninserted into the XhoI site of plasmid pUC-kan1 treated by S1 nucleaseto generate blunt ends; this generated plasmid pUC-kan202 (FIG. 13).This cloning strategy creates an in-frame fusion of the ORF1 gene ofplasmid k1 with the N-10 terminal extremity of the aph[3′]-I gene ofTn903: in the fusion, the first 11 amino acids of the aph[3′]-I geneproduct have been replaced by the first 11 amino acids of ORF1, and theexpression of this hybrid gene is under the control of a K. lactispromoter. The nucleotide sequence surrounding the initation codon of thefusion protein ORF1-APH is as follows (codons originating from ORF1 areunderlined, and the first codons from APH are italicized):5′-TTACATTATTAATTTAAAA ATG GAT TTC AAA GAT AAG GCT TTA AAT GAT CTA AGGCCG CGA TTA AAT TCC AAC . . . -3′

E4.4. Construction and Stability of Plasmid pKan707 in K. lactis.

Plasmid pCXJ1 was cleaved by HindIII, treated with the Klenow fragmentof E. coli DNA polymerase I, then ligated with the 1.2 kb ScaI-HincIIfragment coding for the ORF1-APH fusion expressed under control of theK. lactis P_(k1) promoter deriving from plasmid pUC-Kan202. Theresulting plasmid (pKan707, FIG. 14) confers very high levels ofresistance to G418 (Geneticin, GIBCO, Grand Island, N.Y.) in strains ofK. lactis (>2.5 g/l), is able to transform K. lactis strains cir° due tothe functional integrity of replicon pKD1, can be amplified to 70-100copies per cell, and can be stably maintained in the absence ofselection pressure (FIG. 15). This high stability, coupled with thepresence of a dominant marker permitting the transformation ofindustrial strains of Kluyveromyces, make plasmid pKan707 a highperformance vector for the expression of proteins in yeasts of the genusKluyveromyces.

Example 5 Construction of Expression Plasmids pYG221B (Prepro-HSA) andpYG308B (Prepro-HSA-V1V2)

The SalI restriction fragment coding for the hybrid proteinprepro-HSA-V1V2 expressed under control of the PGK promoter of S.cerevisiae was purified by electrolution from plasmid pYG306 cut by thecorresponding enzyme, and then cloned into the SalI site of plasmidpKan707, to generate plasmids pYG308A and pYG308B which aredistinguished only by the orientation of the SalI fragment in relationto the vector pKan707. A restriction map of plasmid pYG308B is shown inFIG. 16.

Plasmid pYG221B is a control construction coding for prepro-HSA alone;this plasmid was constructed as for plasmid pYG308B (prepro-HSA-V1V2):the SalI fragment coding for prepro-HSA expressed under control of thePGK promoter was purified from plasmid pYG210 and cloned into the SalIsite of plasmid pKan707 to generate plasmid pYG221B (FIG. 17). PlasmidspYG221B (prepro-HSA) and pYG308B (prepro-HSA-V1V2) possess the sameorientation of the SalI expression cassettes in relation to the vectorand are strictly isogenic except for the difference of the MstII-HindIIIfragment located immediately upstream of the PGK terminator. Thenucleotide sequence of the MstII-HindIII fragment in plasmid pYG221B(prepro-HSA) is as follows (the translation stop codon for theprepro-HSA gene is in bold type):5′-CCTTAGGCTTATAACATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAAAGCTT-3′The nucleotide sequence of the MstII-HindIII fragment of plasmid pYG308Bis included in the sequence of the MstII-SmaI fragment shown in FIG. 2.

Example 6 Transformation of Yeasts

Transformation of yeasts of the genus Kluyveromyces and in particular K.lactis strain MW98-8C, was performed by treating whole cells withlithium acetate (Ito H. et al., J. Bacteriol. 153 (1983) 163-168),adapted as follows. Cells were grown in shaker flasks in 50 ml of YPDmedium at 28° C., until reaching an optical density of 0.6-0.8, at whichtime they were harvested by low speed centrifugation, washed in sterileTE (10 mM Tris HCl pH 7.4; 1 mM EDTA), resuspended in 3-4 ml of lithiumacetate (0.1 M in TE) to give a cell density of 2×10⁸ cells/ml, thenincubated 1 hour at 30° C. with moderate agitation. Aliquots of 0.1 mlof the resulting suspension of competent cells were incubated 1 hour at30° C. in the presence of DNA and polyethylene glycol (PEG₄₀₀₀, Sigma)at a final concentration of 35%. After a 5 minute thermal shock at 42°C., cells were washed twice, resuspended in 0.2 ml sterile water, andincubated 16 hours at 28° C. in 2 ml YPD to allow for phenotypicexpression of the ORF1-APH fusion protein expressed under control ofpromoter P_(k1); 200 μl of the resulting cell suspension were spread onYPD selective plates (G418, 200 μg/ml). Plates were incubated at 28° C.and transformants appeared after 2 to 3 days growth.

Example 7 Secretion of Albumin and its Variants by Yeasts of the GenusKluyveromyces

After selection on rich medium supplemented with G418, recombinantclones were tested for their capacity to secrete the mature form ofalbumin or the hybrid protein HSA-V1V2. Certain clones corresponding tostrain MW98-8C transformed by plasmids pYG221B (prepro-HSA) or pYG308B(prepro-HSA-V1V2) were incubated in selective liquid rich medium at 28°C. Culture supernatants were prepared by centrifugation when cellsreached stationary phase, then concentrated by precipitation with 60%ethanol for 30 minutes at 20° C. Supernatants were tested afterelectrophoresis through 8.5% polyacrylamide gels, either by directCoomassie blue staining of the gel (FIG. 18, panel A), or byimmunoblotting using as primary antibody a rabbit polyclonal anti-HSAserum (FIG. 18, panel B) or a rabbit polyclonal anti-CD4 serum (FIG. 18,panel C). For immunoblot experiments, the nitrocellulose filter wasfirst incubated in the presence of specific rabbit antibodies, thenwashed several times, incubated with a biotinylated goat anti-rabbitIg's serum, then incubated in the presence of an avidin-peroxidasecomplex using the “ABC” kit distributed by Vectastain (Biosys S. A.,Compiègne, France). The immunologic reaction was then revealed byaddition of diamino-3,3′ benzidine tetrachlorydrate (Prolabo) in thepresence of oxygenated water, according to the kit recommendations. Theresults shown in FIG. 18 demonstrate that the hybrid protein HSA-V1V2 isrecognized by both the anti-HSA antibodies and the anti-CD4 antibodies,whereas HSA is only recognized by the anti-HSA antibodies.

Example 8 Purification and Molecular Characterization of SecretedProducts

After ethanol precipitation of the culture supernatants corresponding tothe K. lactis strain MW98-8C transformed by plasmids pYG221B(prepro-HSA) and pYG308B (prepro-HSA-V1V2), the pellet was resolubilizedin a 50 mM Tris-HCl buffer, pH 8.0. The HSA-CD4 and HSA proteins werepurified by affinity chromatography on Trisacryl-Blue (IBF). Anadditional purification by ion exchange chromatography can be performedif necessary. After elution, protein-containing fractions were combined,dialyzed against water and lyophylized before being characterized.Sequencing (Applied Biosystem) of the hybrid protein secreted by K.lactis strain MW98-8C revealed the expected N-terminal sequence ofalbumin (Asp-Ala-His . . . ), demonstrating the proper maturation of theprotein.

The isoelectric point was determined by isoelectrofocalization to be 5.5for the HSA-V1V2 protein and 4.8 for HSA.

The HSA-V1V2 protein is recognized by the monoclonal mouse antibodiesOKT4A and Leu3A directed against human CD4, as well as by a polyclonalanti-HSA serum (FIG. 19), and can be assayed by the ELISA method(Enzyme-Linked Immuno-Sorbent Assay, FIG. 20). The substrate for theperoxidase used in these two experiments is2-2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt(ABTS) (Fluka, Switzerland).

Example 9 Characterization of the Anti-Viral Properties of the HSA-CD4Variants

The proteins corresponding to albumin (negative control) and to theHSA-V1V2 fusion purified from culture supernatants of K. lactis strainMW98-8C transformed respectively by plasmids pYG221B (prepro-HSA) andpYG308B (prepro-HSA-V1V2) as in examples 7 and 8, were tested in vitrofor antiviral activity and compared to the entire soluble CD4 moleculepurified from CHO (Chinese Hamster Ovary) cells. Protein concentrationsare expressed in molarity and were determined both by methods to measureproteins in solution as well as by comparison of successive dilutions ofeach protein after electrophoretic migration in polyacrylamide gelsfollowed by silver nitrate staining.

FIG. 21 shows that the HSA-V1V2 fusion is able to inhibit in vitro thebinding of the viral glycoprotein gp160 (uncleaved precursor of gp120)to the CD4 receptor in soluble phase. In this experiment, the ELISAplates were covered with purified recombinant CD4 and incubated withrecombinant gp160 (125 femtomoles) and having been preincubated withvarying quantities of CD4, albumin, or the hybrid protein HSA-V1V2. Theresidual binding of gp160 to CD4 was then revealed by the successiveaddition of mouse monoclonal anti-gp160 (110.4), followed by the bindingof a goat serum linked to peroxidase and directed against mouseantibodies. After addition of a chromogenic substrate(orthophenyldialenine) in the presence of oxygenated water, opticaldensity was measured at 492 nm. The results reported in FIG. 21demonstrate that the hybrid protein HSA-V1V2 is able to inhibit thebinding of gp160 to CD4 in soluble phase, in a manner indistinguishablefrom the positive control corresponding to the entire CD4 molecule. Incontrast, the albumin molecule is almost completely inactive in thisregard. This experiment indicates that the inhibition by the hybridprotein is due to the presence of the V1V2 domains in a conformation andaccessibility similar to the complete CD4 receptor.

FIG. 22 shows that the HSA-V1V2 hybrid is able to inhibit the in vitrobinding of the HIV-1 virus to cells expressing the CD4 receptor on theirmembranes. In this experiment, a cell line that expresses highquantities of CD4 receptor (lymphoblastic cell line CEM13) was incubatedwith 2 μg of heat-inactivated viral particles that had been preincubatedwith 116 picomoles of either HSA-V1V2 (10.7 μg), HSA (7.5 μg), orrecombinant entire CD4 purified from CHO cells (5 μg). The binding ofthe inactivated viral particles to cell membranes was revealed bysuccessive incubations of a mouse monoclonal anti-gp120 antibody and agoat anti-mouse IgG serum marked with phycoerythrin. The negativecontrol corresponds to cell line CEM13 incubated successively with thesetwo antibodies. Fluorescence was measured with a cell sorter (FIG. 22,panel A) and the results are presented in the form of a histogram (FIG.22, panel B). This experiment shows that the HSA-V1V2 protein is able toinhibit the binding of the HIV-1 virus to CEM13 cells almost completely.Furthermore, this inhibition is slightly higher than that of thecomplete CD4 molecule; this can be explained by the fact that albumin,known for its adhesive properties, is able to inhibit the binding of thevirus to the target cells in a nonspecific manner and with a lowefficiency.

The HSA-CD4 protein is also able to inhibit viral infection ofpermissive cells in cell culture. This inhibition was measured either byassaying the production of viral antigens (viral p24) using the kitELAVIA-AG1 (Diagnostics Pasteur), or the kit p24-ELISA (Dupont), or bymeasuring the reverse transcriptase activity by the technique ofSchwartz et al. (Aids Research and Human Retroviruses 4 (1988) 441-448).The experimental protocol was as follows: the product of interest at afinal concentration X was first preincubated with supernatants of CEM13cells infected by the isolate LAV-Bru1 of virus HIV-1 (dilution 1/250,1/2500 and 1/25000) in a total volume of 1 ml of culture medium (RPMI1640 containing 10% fetal calf serum, 1% L-glutamine and 1%penicillin-streptomycin-neomycin). The mixture was then transfered ontoa pellet of 5×10⁵ permissive cells (e.g. MT2, CEM13, or H9) andincubated in tubes for 2 hours at 37° C. for infection to occur. Theinfection could also be carried out on microtitration plates with 10⁴cells per well in 100 μl of complete medium. A volume of 100 μl of thevirus that had been preincubated with the product to be tested was thenadded, followed by 50 μl of the product at 5× concentration. Cells werethen washed twice with 5 ml RPMI 1640 and resuspended in culture mediumat a density of 2:5×10⁵ cells/ml. 100 μl of this suspension was thenaliquoted into each well of microtitration plates which already contain100 μl of the product at 2× concentration, and the plates were incubatedat 37° C. in a humid atmosphere containing 5% CO₂. At different days(D3-D4-D6-D8-D10-D12-D14-D16-D19-D21 and D25), 100 μl of supernatant wasremoved and the p24 viral production as well as the reversetranscriptase activity were assayed. Cells were then resuspended anddistributed onto microtitration plates for assays of cell viability(MTT) as described above. To the 50 μl remaining on the original plates,200 μl of culture medium containing the product to be tested atconcentration X were added, and infection was allowed to progress untilthe next sampling. For the cell viability test, 10 μl of MTT at 5 mg/mlfiltered on 0.2 μm filters was added to each well and plates wereincubated 4 hours at 37° C. in a humid atmosphere containing 5% CO₂.Then to each well was added 150 μl of an isopropanol/0.04 N HCl mixture,and the Formazan crystals were resuspended. Optical density from 520 to570 nm was measured on a Titertek plate reader; this measure reflectscell viability (Schwartz et al., Aids Research and Human Retroviruses 4(1988) 441-448).

FIG. 23 shows an example of inhibition of infectivity in cell culture(cell line CEM13) as measured by reverse transcriptase activity. Thisdemonstrates that the HSA-V1V2 hybrid is able to reduce the infectivityof the HIV-1 virus to the same extent as the soluble CD4 molecule.

Example 10 Stability of the Hybrid Proteins In Vivo

It has been shown that first generation soluble CD4 possesses ahalf-life of 20 minutes in rabbits (Capon D. J. et al.; Nature 337(1989) 525-531). We have therefore compared the half-life in rabbits ofthe HSA-CD4 hybrid to soluble CD4 and to recombinant HSA produced inyeast and purified in the same manner as HSA-CD4. In these experiments,at least 2 male NZW(Hy/Cr) rabbits weighing 2.5-2.8 kg were used foreach product. Rabbits were kept in a room maintained at a temperature of18.5-20.5° C. and a humidity of 45-65%, with 13 hours light/day. Eachproduct was administered in a single injection lasting 10 seconds in themarginal vein of the ear. The same molar quantity of each product wasinjected: 250 μg of CD4 per rabbit, 400 μg of HSA per rabbit, or 500 μgof HSA-CD4 per rabbit, in 1 ml physiologic serum. Three to four ml bloodsamples were taken, mixed with lithium heparinate and centrifuged 15 minat 3500 rpm; samples were then divided into three aliquots, rapidlyfrozen at −20° C., then assayed by an ELISA method. Blood samples fromrabbits injected with CD4 were taken before injection (To), then 5 min,10 min, 20 min, 30 min, 1 h, 2 h, 4 h and 8 h after injection. Bloodsamples from rabbits injected with HSA-CD4 or HSA were taken at To, 30min, 1 h, 2 h, 4 h, 8 h, 24 h, 32 h, 48 h, 56 h, 72 h, 80 h, 96 h, 104 hand 168 h after injection.

Assays of the CD4 molecule were carried out on Dynatech M129Bmicrotitration plates previously covered with the HSA-CD4 hybridprotein. Increasing concentrations of CD4 or the samples to be assayedwere then added in the presence of the mouse monoclonal antibody OKT4A(Ortho-Diagnostic, dilution 1/1000); after incubation and washing of theplates, the residual binding of antibody OKT4A was revealed by additionof antibodies coupled to peroxidase (Nordic, dilution 1/1000) anddirected against mouse IgG. Measurements were made at OD 405 nm in thepresence of the peroxidase substrate ABTS (Fluka).

Assays of recombinant HSA were carried out on Dynatech M129Bmicrotitration plates previously covered with anti-HSA serum (Sigma Ref.A0659, dilution 1/1000); increasing concentrations of HSA or samples tobe measured were then added, followed by addition of anti-HSA serumcoupled to peroxidase (Nordic, dilution 1/1000). Measurements were madeat OD 405 nm as above.

Two different assays were done for the HSA-CD4 hybrid: either the assayfor the HSA moiety alone, using the same methods as for recombinant HSA,or an assay for the HSA moiety coupled with an assay for the CD4 moiety.In the latter case, microtitration plates were covered first withanti-HSA serum (Sigma Ref. A0659, dilution 1/1000), then incubated withthe samples to be assayed. The mouse monoclonal antibody Leu3A directedagainst CD4 was then added, followed by antibodies coupled to peroxidase(Nordic, dilution 1/1000) and directed against mouse antibodies.Measurements were made at 405 nm as described above.

The curves for each of these assays are given in FIG. 24. Interpretationof these results allows the evaluation of the pharmacokineticcharacteristics of each product in the rabbit. The half-lives measuredfor each product are as follows: CD4: 0.25 ± 0.1 h HSA: 47 ± 6 hHSA-CD4: 34 ± 4 h

These results underscore the following points:

-   -   1/ The coupling of CD4 to albumin allows a significant increase        in the stability of CD4 in the organism since the half-life of        elimination is increased 140-fold.    -   2/ The half-life of elimination of the HSA-CD4 hybrid is        comparable to that of HSA.    -   3/ The clearance of CD4 is approximately 3 ml/min/kg while that        of HSA and HSA-CD4 is approximately 0.02 ml/min/kg.    -   4/ The CD4 moiety of the HSA-CD4 hybrid apparently retains an        active conformation (i.e. able to bind gp120) since the assay        for CD4 involves the Leu3A monoclonal antibody which recognizes        an epitope close to the binding site of gp120 (Sattentau Q. J.        et al., Science 234 (1986) 1120-1123; Peterson A. and Seed B.,        Cell 54 (1988) 65-72). Furthermore, the two independent assay        methods for the HSA-CD4 hybrid gave essentially the same result,        which suggests that the CD4 moiety is not preferentially        degraded in vivo.

It is noteworthy that the volume of distribution of HSA and HSA-CD4 isclose to that of the blood compartment, and therefore suggests adistribution of the product limited to the extracellular compartment.

Example 11 Generic Constructions of the Type HSA-CD4

E.11.1. Introduction of AhaII and BglII Sites at the End of the PreproRegion of HSA.

Introduction of the AhaII restriction site was carried out bysite-directed mutagenesis using plasmid pYG232 and oligodeoxynucleotideSq1187, to generate plasmid pYG364. Plasmid pYG232 was obtained bycloning the HindIII fragment coding for prepro-HSA into the vectorM13mp9. The sequence of oligodeoxynucleotide Sq1187 is (the AhaII siteis in bold type): 5′-GTGTTTCGTCGAGACGCCCACAAGAGTGAGG-3′.It should be noted that creation of the AhaII site does not modify theprotein sequence of the N-terminal of mature HSA. The construction ofplasmid pYG364 is shown in FIG. 25.

Plasmid pYG233 was obtained in analogous fashion, after site-directedmutagenesis of plasmid pYG232 using oligodeoxynucleotide Sq648 (thecodons specificying the amino acid pair Arg-Arg situated at the end ofthe prepro region of HSA are in bold type, and the BglII site isunderlined): 5′-GGTGTGTTTCGT AGATCTGCACACAAGAGTGAGG-3′The creation of this restriction site does not change the proteinsequence of the prepro region of HSA. In contrast, the first amino acidof the mature protein is changed from an aspartate to a serine; plasmidpYG233 therefore codes for a mature HSA modified at its N-terminal(HSA*, FIG. 25).

E11.2. Introduction of the Prepro Region of HSA Upstream of the CD4Receptor.

The introduction of the prepro region of HSA upstream of the V1V2domains of the CD4 receptor was accomplished by site-directedmutagenesis, to generate plasmid pYG347 as shown in FIGS. 26 and 27.Plasmid pYG231 (FIG. 26) is an intermediate construction correspondingto a pUC-type replicon into which has been cloned a SalI fragmentcarrying the expression cassette for HSA (yeast promoter/prepro-HSA/PGKterminator of S. cerevisiae). Plasmid pYG234 is isogenic to plasmidpYG231 except that oligodeoxynucleotide Sq648 was used to carry out thein vitro mutagenesis (E.11.1.).

Plasmid pYG347 was obtained by site-directed mutagenesis of plasmidpYG332 with oligodeoxynucleotide Sq1092 (FIG. 27) whose sequence is asfollows (HSA sequence is in italics and CD4 sequence is in bold type):5′-CCAGGGGTGTGTTTCGTCGA AAGAAAGTGGTGCTGGGC-3′

Plasmid pYG347 therefore carries a HindIII fragment composed of the 21nucleotides preceding the ATG codon of the PGK gene of S. cerevisiae,the ATG translation initiation codon, and the prepro region of HSA(LP_(HSA)) immediately followed by the V1V2 domains of the CD4 receptor.

E.11.3. Introduction of an AhaII Site at the End of the V1 Domain of theCD4 Receptor.

The introduction of an AhaII site at the end of the V1 domain of the CD4receptor was accomplished by site-directed mutagenesis usingoligo-deoxynucleotide Sq1185 and a derivative of plasmid pYG347 (pYG368,FIG. 28), to generate plasmid pYG362. The sequence ofoligodeoxynucleotide Sq1185 is (the AhaII site is shown in bold type):5′-CCAACTCTGACACCGACGCCCACCTGCTTCAGG-3′.

Plasmid pYG362 therefore carries a HindIII-AhaII fragment composed ofthe 21 nucleotides preceding the ATG codon of the PGK gene of S.cerevisiae followed by the coding sequence of the HSA prepro regionfused to the V1 domain of the CD4 receptor, according to example E.11.2.In a fusion such as the example given here, the V1 domain of the CD4receptor carries 106 amino acids and includes the functional bindingsite of the HIV-1 viral glycoprotein gp120.

E.11.4. Introduction of an AhaII Site at the End of the V2 Domain of theCD4 Receptor.

The introduction of an AhaII site at the end of the V2 domain of the CD4receptor was accomplished by site-directed mutagenesis usingoligo-deoxynucleotide Sq1186 and plasmid pYG368, to generate plasmidpYG363 (FIG. 28). The sequence of oligodeoxynucleotide Sq1186 is (theAhaII site is shown in bold type): 5′-GCTAGCTTTCGACGCCGGGGGAATTCG-3′.Plasmid pYG363 therefore carries a HindIII-AhaII fragment composed ofthe 21 nucleotides preceding the ATG codon of the PGK gene of S.cerevisiae followed by the coding sequence for the HSA prepro regionfused to the V1V2 domains of the CD4 receptor. In this particularfusion, the V1V2 domains contain 179 amino acids.

Other variants of plasmid pYG363 were generated by site-directedmutagenesis in order to introduce an AhaII at different places in the V2domain of the CD4 receptor. In particular, plasmid pYG511, shown in FIG.28, does not contain the amino acid pair Lys-Lys at positions 166-167 ofthe V2 domain; this is due to the oligodeoxynucleotide used (Sq1252; theAhaII site is shown in bold type):5′-GCAGAACCAGAAGGACGCCAAGGTGGAGTTC-3′.

E11.5. Generic Constructions of the Type V1-HSA.

The plasmids described in the preceding examples allow for thegeneration of HindIII restriction fragments coding for hybrid proteinsin which the receptor of the HIV-1 virus (fused to the signal sequenceof HSA) precedes HSA. For example, plasmids pYG362 and PYG364 arerespectively the source of a HindIII-AhaII fragment (fusion of the HSAprepro region to the V1 domain of the CD4 receptor), and an AhaII-NcoIfragment (N-terminal region of mature HSA obtained as in exampleE.11.1.). The ligation of these fragments with the NcoI-KpnI fragment(C-terminal region of HSA and terminator of the PGK gene of S.cerevisiae) in an analogue of plasmid pYG18 cut by HindIII and KpnIgenerates plasmid pYG371 whose structure is shown in FIG. 29. In thisplasmid, the gene coding for the hybrid protein V1-HSA fused to the HSAprepro region is cloned into an expression cassette functional inyeasts. This cassette can then be cloned into a replicative vector thatcan be selected in yeasts, for example the vector pKan707, whichgenerates expression plasmid pYG373B (FIG. 30).

E11.6. Generic Constructions of the Type V1V2-HSA.

Hybrid proteins of the type V1V2-HSA were generated by the followingstrategy: in a first step, plasmids pYG511 (FIG. 28) and pYG374 (FIG.29) were respectively the source of the restriction fragmentsBglII-AhaII (fusion of the HSA prepro region and the V1V2 domains of theCD4 receptor) and AhaII-KpnI (in-frame fusion between mature HSA and theV1V2 domains of the CD4 receptor as exemplified in E.12.2). Ligation ofthese fragments in a chloramphenicol resistant derivative of pBluescriptII SK(+) vector (plasmid pSCBK(+), Stratagene) generates plasmid pYG537(FIG. 31). This plasmid contains a HindIII fragment coding for thehybrid bivalent molecule CD4HSA-CD4 fused in-frame with the signalpeptide of HSA as exemplified in E.11.2. Plasmid pYG547 which contains aHindIII fragment coding for the hybrid protein V1V2-HSA fused in-framewith the prepro region of HSA, was then derived by substitution of thePstI-KpnI fragment of pYG537 by the PstI-KpnI fragment from plasmidpYG371. The HindIII fragment carried by plasmid pYGS47 can then beexpressed under control of a functional yeast promoter cloned in avector that replicates, for example, in yeasts of the genusKluyveromyces. One example is the expression plasmid pYG560 whosestructure and restriction map are shown in FIG. 32. Vector pYG105 usedin this particular example corresponds to plasmid pKan707 whose HindIIIsite has been destroyed by site-directed mutagenesis(oligodeoxynucleotide Sq1053, 5′-GAAATGCATAAGCTCTTGCCATTCTCACCG-3′) andwhose SaI-SacI fragment coding for the URA3 gene has been replaced by aSalI-SacI fragment carrying a cassette made up of a promoter, aterminator, and a unique HindIII site.

Example 12 Bivalent Hybrid Protein Complexes

E.12.1. Introduction of a Stop Codon Downstream of the V1 Domain of theCD4 Receptor

Conventional techniques permit the introduction of a translation stopcodon downstream of the domain of the CD4 receptor which is responsiblefor the binding of the HIV-1 viral glycoprotein gp120. For example, aTAA codon, immediately followed by a HindIII site, was introduced bysite-directed mutagenesis downstream of the V1 domain of the CD4receptor. In particular, the TAA codon was placed immediately after theamino acid in position 106 of the CD4 receptor (Thr¹⁰⁶) usingoligodeoxynucleotide Sq1034 and a plasmid analogous to plasmid M13-CD4as matrix. The sequence of oligodeoxynucleotide Sq1034 is (the stopcodon and the HindIII site are in bold type):5′-ACTGCCAACTCTGACACCTAAAAGCTTGGATCCCACCTGCTTCAGGG GCAG-3′

E12.2 Constructions of the Type CD4HSA-CD4.

The plasmids described in examples E.11.5. et E.11.6. which exemplifygeneric constructions of the type CD4-HSA allow for the easy generationof bivalent constructions of the type CD4-HSA-CD4. Plasmids pYG374(V1-HSA-V1V2) or pYG375 (V1-HSA-V1) illustrate two of these genericconstructions: for example, the small MstII-HindIII fragment of plasmidpYG371 which codes for the last amino acids of HSA can be replaced bythe MstII-HindIII fragment coding for the last 3 amino acids of HSAfused to the V1V2 domains of the CD4 receptor (plasmid pYG374, FIG. 29),or to the V1 domain alone (plasmid pYG375, FIG. 29). The genes codingfor such bivalent hybrid proteins can then be expressed under control ofa functional yeast promoter that replicates, for example, in yeasts ofthe genus Kluyveromyces. Examples of such expression plasmids are theplasmids pYG380B (V1-HSA-V1V2) and pYG381B (V1-HSA-V1) which arestrictly isogenic to plasmid pYG373B (V1-HSA) except for the structuralgenes encoded in the HindIII fragments. The bivalent hybrid proteinsdescribed here are expressed at levels comparable to their monovalentequivalents, indicating a very weak level of recombination of therepeated sequences resulting from genetic recombination in vivo (FIG.33).

The construction of HindIII fragments coding for bivalent hybridproteins of the type V1V2-HSA-V1V2 has already been described in FIG. 31(plasmid pYG537). The genes coding for such bivalent hybrid proteins ofthe type CD4HSA-CD4 can then be expressed under control of a functionalyeast promoter in a vector that replicates, for example, in yeasts ofthe genus Kluyveromyces. Such expression plasmids are generated by thestrategy described in FIG. 32 (cloning of a HindIII fragment intoplasmids analogous to plasmid pYG560).

E.12.3. Introduction of a Dimerization Domain.

For a given hybrid protein derived from albumin and carrying one orseveral binding sites for the HIV-1 virus, it may be desirable toinclude a polypeptide conferring a dimerization function, which allowsfor the agglomeration of trapped virus particles. An example of such adimerization function is the “Leucine Zipper” (LZ) domain present incertain transcription regulatory proteins (JUN, FOS . . . ). Inparticular, it is possible to generate a BglII-AhaII fragment coding,for example, for the LZ of JUN, by the PCR technique by using thefollowing oligodeoxynucleotides and the plasmid pTS301 (which codes foran in-frame fusion between the bacterial protein LexA and the LZ of JUN,T. Schmidt and M. Schnar, unpublished results) as matrix (BglII andAhaII sites are underlined):5′-GGTAGGTCGTGTGGACGCCAGATCTTTGGAAAGAATTGCCCGTCTGG AAG-3′5′-CTGCAGGTTAGGCGTCGCCAACCAGTTGCTTCAGCTGTGC-3′

This BglII-AhaII fragment (FIG. 34) can be ligated to the HindIII-BglIIfragment of plasmid pYG233 (HSA prepro region, FIG. 25) and theAhaII-HindIII fragment as shown in one of the examples E.11. to generatea HindIII fragment coding for hybrid proteins of the type LZ-HSA-CD4,fused to the signal sequence of HSA. To prevent a possible dimerizationof these molecules during their transit through the yeast secretorypathway, it may be desirable to utilize a LZ domain which cannot formhomodimers. In this case the “Leucine Zipper” of FOS is preferred;dimerization would then result when these proteins are placed in thepresence of other hybrid proteins carrying the LZ of JUN.

The introduction of carefully selected restriction sites that permit theconstruction of genes coding for hybrid proteins of the type LZ-CD4-HSAor LZ-CD4-HSA-CD4 is also possible, using conventional in vitromutagenesis techniques or by PCR.

Example 13 Genetic Engineering of the Hinge Region Between the CD4 andHSA Moieties

E-13.1. Strategy Using Bal31 Exonuclease.

Proteins secreted by strain MW98-8C transformed by expression plasmidsfor HSA-CD4 hybrid proteins in which the CD4 moiety is carried on theMstII-HindIII fragment in the natural MstII site of HSA (plasmid pYG308Bfor example), were analyzed. FIG. 35 demonstrates the presence of atleast two cleavage products comigrating with the albumin standard (panel2), which have a mature HSA N-terminal sequence, and which are notdetectabe using polyclonal antibodies directed against human CD4 (panels2 and 3). It is shown that these cleavage products are generated duringtransit through the yeast secretory pathway, probably by the KEX1 enzymeof K. lactis (or another protease with a specificity analogous to theendoprotease YAP3 of S. cerevisiae whose gene has been cloned andsequenced (Egel-Mitani M. et al. Yeast 6 (1990) 127-137). Therefore, thepeptide environment of the hinge region between the HSA and CD4 moietieswas modified, notably by fusion of the CD4 molecule (or one of itsvariants capable of binding the gp120 protein of HIV-1) to HSAN-terminal regions of varying length, according to the followingstrategy: plasmid pYG400 is an intermediate plasmid carrying theprepro-HSA gene, optimized with respect to the nucleotide sequenceupstream of the ATG codon, on a HindIII fragment. This plasmid waslinearized at its unique MstII site and partially digested by Bal31exonuclease. After inactivation of this enzyme, the reaction mixture wastreated with the Klenow fragment of E. coli DNA polymerase I and thensubjected to ligation in the presence of an equimolar mixture ofoligodeoxynucleotides Sq1462 (5′-GATCCCCTAAGG-3′) and Sq1463(5′-CCTTAGGG-3′) which together form a synthetic adaptor containing aMstII site preceding a BamHI site. After ligation, the reaction mixturewas digested with HindIII and BamHI and fragments between 0.7 and 2.0 kbin size were separated by electroelution and cloned into an M13mp19vector cut by the same enzymes. 10⁶ lytic plaques were thus obtained ofwhich approximately one-third gave a blue color in the presence of IPTGand XGAL. Phage clones which remained blue were then sequenced, and inmost cases contained an in-frame fusion between the HSA N-terminalmoiety and β-galactosidase. These composite genes therefore containHindIII-MstII fragments carrying sections of the N-terminal of HSA; FIG.36 shows several examples among the C-terminal two-thirds of HSA. Thesefragments were then ligated with a MstII-HindIII fragment correspondingto the CD4 moiety (for example the V1V2 domains of FIG. 2, or the V1domain alone), which generates HindIII fragments coding for hybridproteins of the type HSA-CD4 in which the HSA moiety is of varyinglength. These restriction fragments were then cloned in the properorientation into an expression cassette carrying a yeast promoter andterminator, and the assembly was introduced into yeasts. After growth ofthe culture, the hybrid proteins HSA-CD4 can be obtained in the culturemedium; certain of these hybrids have an increased resistance toproteolytic cleavage in the hinge region (FIG. 35).

E.13.2. Mutation of Dibasic Amino Acid Pairs.

Another way to prevent cleavage by endoproteases with specificity fordibasic amino acid pairs is to suppress these sites in the area of thehinge region between the HSA and the CD4 moieties (FIG. 37), or in thearea of the hinge region between CD4 and HSA (FIG. 38). As an example,the hinge region present in the hybrid protein HSA-V1V2 coded by plasmidpYG308B is represented in FIG. 37 (panel 1), and points out the presenceof a Lys-Lys pair in the C-terminal of HSA and two such pairs in theN-terminal of the V1 domain of CD4. Using site-directed mutagenesis,these potential endoprotease cleavage sites can be suppressed bychanging the second lysine in each pair to a glutamine (Risler J. L. etal., J. Mol. Biol. 204 (1988) 1019-1029), for example by using plasmidM13-ompA-CD4 as matrix and the oligodeoxynucleotides Sq1090 and Sq1091(the codons specifying glutamine are in bold type):5′-GTGCTGGGCAAACAAGGGGATACAG-3′ 5′-GGCTTAAAGCAAGTGGTGCTG-3′

Plasmid M13-ompA-CD4 is a derivative of plasmid M13-CD4 in which thesignal sequence of the ompA gene of E. coli is fused in frame to the CD4receptor using the MstII site generated by PCR upstream of the V1 domain(example 1).

E13.3. Introduction of a Synthetic Hinge Region.

In order to promote an optimal interaction between the CD4 moiety fusedto HSA, and the gp120 protein of the HIV-1 virus, it may be desirable tocorrectly space the two protein moieties which form the building blocksof the hybrid protein HSA-CD4. For example, a synthetic hinge region canbe created between the HSA and CD4 moieties by site-directed mutagenesisto introduce a fragment of troponin C between amino acids 572 and 582 ofmature HSA (FIG. 37, panel 3). In this particular example, the junctionpeptide was introduced via site-directed mutagenesis by using arecombinant M13 phage (carrying the PstI-SacI fragment coding for thein-frame fusion between the C-terminal portion of HSA and the C-terminalpart of the CD4 receptor) as matrix and oligodeoxynucleotide Sq1445:5′-TGCTTTGCCGAGGAGGGTAAGGAAGACGCTAAGGG-TAAGTCTGAAGAAGAAGCCTTAGGCTTAAAGAAA-3′.

Similar techniques also permit the introduction of such a synthetichinge region between the HSA and CD4 moieties (junction peptide, FIG.38, panel 3).

Example 14 Expression of Hybrid Proteins Under the Control of DifferentPromoters

For a given protein secreted by cells at high levels, there exists athreshold above which the level of expression is incompatible with cellsurvival. Hence there exist certain combinations of secreted protein,promoter utilized to control its expression, and genetic background thatare optimal for the most efficient and least costly production. It istherefore important to be able to express the hybrid proteins which arethe object of the present invention under the control of variouspromoters. The composite genes coding for these proteins are generallycarried on a HindIII restriction fragment that can be cloned in theproper orientation into the HindIII site of a functional expressioncassette of vectors that replicate in yeasts. The expression cassettecan contain promoters that allow for constitutive or regulatedexpression of the hybrid protein, depending on the level of expressiondesired. Examples of plasmids with these characteristics include plasmidpYG105 (LAC4 promoter of K. lactis, FIG. 32), plasmid pYG106 (PGKpromoter of S. cerevisiae), or plasmid pYG536 (PHO5 promoter of S.cerevisiae) etc . . . In addition, hybrid promoters can be used in whichthe UAS regions of tightly regulated promoters have been added, such asthe hybrid promoters carried by plasmids pYG44 (PGK/LAC hybrid, Europeanpatent application EP No 89 10480), pYG373B (PGK/GAL hybrid), pYG258(PHO5/LAC hybrid) etc. . . .

1/ Hybrid macromolecule characterized by the fact that it carries eitherthe active domain of a receptor for a given virus, or the active domainof a molecule which can bind to the virus, or the active domain of areceptor of a ligand intervening in a pathological process, coupled toalbumin or a variant of albumin. 2/ Macromolecule according to claim 1,in which the receptor is a membrane receptor. 3/ macromolecule accordingto claims 1 and 2, characterized by the fact that such a macromoleculeis substantially proteinic. 4/ Macromolecule according to claims 1, 2 or3, in which the coupling is covalent. 5/ Macromolecule according toclaim 4, in which the covalent coupling is accomplished by a peptidelinkage. 6/ Macromolecule according to claims 1, 2, 3, 4 or 5, in whichthe active domain of the receptor is the active domain of a receptornormally used by a virus for its propagation in the host organism. 7/Macromolecule according to claims 1, 2, 3, 4 or 5, in which the activedomain of the receptor is the active domain of a receptor intervening inthe internalization of infectious virions complexed to immunoglobulins.8/ Macromolecule according to claim 7, in which the active domain of thereceptor is the active domain of a receptor of the type FcγRIII. 9/Macromolecule according to claim 8, in which the active domain of thereceptor is the active domain of the receptor CD16. 10/ Macromoleculeaccording to claim 1, 2, 3, 4 or 5 in which the active domain of thereceptor is the active domain of a receptor of a factor intervening inan oncogenic process. 11/ Macromolecule according to claim 10, in whichthe active domain of the receptor is the active domain of a tyrosinekinase-type receptor. 12/ Macromolecule according to claim 11, in whichthe active domain of the receptor is the active domain of theproto-oncogene c-erbB-2. 13/ Macromolecule according to one of theclaims 1 through 12, characterized by the fact that the albumin used isof human origin. 14/ Macromolecule according to one of the claims 1through 6, in which the receptor is all or part of the CD4 molecule usedby the HIV-1 virus for its propagation in the host organism, includingall artificial variations of the region of interaction with the viruswhich have a higher than normal molecular affinity for the virus. 15/Macromolecule according to claim 14 in which the receptor is made up ofthe V1 and V2 domains of the CD4 molecule. 16/ Macromolecule accordingto one of the claims 1 through 15, characterized by the fact that itcarries more than one active domain of the receptor or of the moleculecapable of binding the ligand. 17/ Macromolecule according to one of theclaims 1 through 16, in which albumin or the variant of albumin islocalized at the N-terminal end. 18/ Macromolecule according to claim 17in which a dimerization or polymerization function is incorporated topermit an increase in the local concentration of the active domain ofthe receptor of the virus or of the receptor of the ligand associatedwith an oncogenic process. 19/ Macromolecule according to claims 1 to 18characterized in that it is devoid of proteolytic cleavage sites betweenthe active domain of the receptor or of the molecule capable of bindingsaid ligands, and albumin or said variant of albumin. 20/ Macromoleculeaccording to one of the claims 1 through 19, characterized by the factthat it is obtained by cultivating cells that have been transformed,transfected, or infected by a vector expressing such macromolecule. 21/Macromolecule according to claim 20, in which the transformed cell is ayeast. 22/ Macromolecule according to claim 21, in which the yeast is astrain of the genus Kluyveromyces. 23/ Macromolecule according to claim21, in which the vector is an expression vector derived from plasmidpKD1 in which the genes A, B and C, the origin of replication, and theinverse repeats have been conserved. 24/ A macromolecule according toone of the claims 1 through 23, for use as a pharmaceutical. 25/ For useas a pharmaceutical according to claim 24, a macromolecule composed ofhuman albumin or an albumin variant, and the V1 domain of the CD4molecule. 26/ For use as a pharmaceutical according to claim 25, amacromolecule composed of human albumin or an albumin variant, and theV1V2 domains of CD4. 27/ Cells that have been transformed, transfected,or infected by a vector expressing a macromolecule according to one ofthe claims 1 through
 19. 28/ Cells according the claim 27, characterizedby the fact that these cells are yeasts. 29/ Cells according to claim28, characterized by the fact that the yeast is of the genusKluyveromyces.